Virology tidbits

Virology tidbits

Wednesday, 21 January 2015

Porcine Respiratory Syndrome Virus (PRRSV) and autophagy: a link between the ER stress response and p53?

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus with a positive strand ssRNA genome of approx. 15kB in length, encoding for ten open reading frames (ORFs).
Similar to the genome of the Coronaviridae, the PRRSV genome ORF1a and 1b genes encode for a RNA-dependent RNA Polymerase as well as for a number of non-structural proteins (nsp; 12 in total) which are generated by autoproteolytic cleavage by a virally encoded cysteine protease (nsp2), 3C-like serine protease (nsp4), and papain-like cysteine protease nsp1α/1β. As it is the case for the Coronaviridae, the remaining nsp’s of ORF1 encode for enzymes required for the replication of the viral genome, including a viral RNA Helicase (nsp-10) and endonuclease (nsp-11).
The remaining ORFs 2-7 encode for ORF2a (GP2a), ORF2b (E), ORF3 (GP3), ORF4 (GP4), ORF5a (GP5a), ORF5b (GP5b), ORF6 (M), and ORF7, the viral Nucleocapsid (N) which akin to the coronaviral N protein localises to the nucleolus and is phosphorylated.

PRRSV virus

PRRSV genome: ORFs and nsp's

In PRRSV infected MARC-145 cells, the viral nsp-2 protein is localised in the perinuclear region resembling a localisation at the ER, akin to the coronaviral proteases, thus suggesting that the expression of of nsp-2 may induce autophagy and a ER Stress response as described for the PLP2 and PLpro proteins derived from CoV. Indeed, PRRSV nsp-2 has been described to contain a DUB domain as well as inhibiting the Interferon response although an interaction with STING has not been demonstrated yet.  Although the data for the intracellular localisation of PRRSV structural proteins are incomplete, in the case of the related Equine Arterivirus (EAV), in BHK-21 cells the EAV E protein (encoded by ORF 2a) predominantly localises to the ER and to a lesser degree to the Golgi complex, whereas the viral GL  localises to the Golgi.

As described before for other positive strand RNA viruses such as Chikungunya Virus, Measles Virus, Coxsackie B Virus, Coronavirus as well as EMCV, the expression of viral proteins induces the formation of replication transcription centers (RTCs), double membrane  vesicles (DMV) which contain the viral RNA (both ssRNA and dsRNA intermediates) as well as the enzymes required for transcription including the viral RNA dependent RNA Polymerase and RNA Helicase. Commonly the DMV derive from the ER in a process subverting the autophagy pathway. As described before autophagy -which involves the formation of mature autophagosomes that fuse with lysosomes, ultimately leading to the degradation of the proteins localised within the autophagosome. Alternatively, the contents of autophagic vesicles might be secreted or in the case of viral proteins be processed to be displayed by MHC-Class I and MHC-Class II molecules.  As discussed before, viral proteins -sometimes the same which promote autophagy as for instance the CoV nsp-6 protein- not only promote the formation of DMV but also inhibit the formation of mature autophagosomes and/or the fusion of autophagosomes with lysosomes.

In the case of PRRSV, the viral nsp-2. nsp-3, and nsp-5/6/7 proteins have been demonstrated to localise to the ER and the expression of of the nsp-5/6/7 protein induces the formation of GFP-LC3 positive vesicles, indicating the induction of the formation of autophagosomes. Akin to the CoV nsp-6 protein, the expression of nsp-5/6/7 protein in Vero cells has been postulated to inhibit the fusion of the autophagosome with the lysosome. In the case of both nsp-2 and nsp-3 however the formation of autophagosomes has not been demonstrated (to my knowledge) although in MARC-145 cells and porcine pulmonary alveolar macrophages infected with PRRSV, LC3-II positive autophagosomes accumulate 24 hrs p.i. whilst the fusion with the lysosome is inhibited since the application of Chloroquine does not increase the number of GFP-RFP LC3-II positive punctae nor the levels of LC3-II as measured by western blot. Contrary to these results however, the treatment of PRRSV infected MARC-145 cells with Bafilomycin-A1 suggest that at 120 hrs p.i. PRRSV titers are decreased compared to mock treated cells and that the levels of p62/SQSTM-1 in PRRSV infected cells are lower than in non-infected cells. The difference observed might be due to the experimental conditions since Bafilomycin-A treatment lasted for 48 hrs compared to 6 hrs for Chloroquine treatment as well as different virus strains (PRRSV JXwn06 v. VR-2385), so more experiments are needed to address this issue.

Similar to the RTC induced following the infection of BHK-21 cells with EAV, these vesicles contain the viral nsp-2 and N protein although the presence of dsRNA has not been demonstrated to my knowledge. In contrast to EAV infected MEF, the autophagic machinery however is required for PRRSV replication  as viral titers are significantly lower in MARC-145 transfected with shLC3B, siATG7, siBeclin-1 or shATG5, suggesting that PRRSV -in contrast to EAV- does not induce the formation of autophagy-like vesicles via the ERAD pathway but via the induction of the phagophore via the ATG5/ATG7/Beclin-1 pathway; if however EDEMosomes are formed during PRRSV infection remains to be seen. Viral replication can also be induced by treating cells with Rapamycin, thus inhibiting mTORC1 and promoting autophagy, whereas treatment with 3-Methyladenine (3-MA) decreases viral titers.

Interestingly, the infection of MARC-145 with PRRSV strain VR-2385 activates mTORC1 (and thus inhibits autophagy) at early times post infection (6 h p.i.). So far the impact on viral or starvation induced autophagy has not been investigated, but the author these lines suggests that PRRSV inhibits autophagy at early timepoints p.i. whereas at later timepoints the formation of autophagy like vesicles is induced. This hypothesis is supported by results indicating that PIK-K-Akt kinase signalling is modulate by PRRSV in so far as phosphorylated Akt kinase levels increase at earlier timepoints, but decrease at 12 hrs p.i.  . It is however crucial to compare proteins derived from highly virulent strains to those derived from attenuated or less pathogenic strains. 

Induction of p53 and DRAM-1 dependent autophagy via the ER stress by
PRRSV: hypothetical model

PRRSV and the ER stress response: does nsp-2, nsp-4, or nsp-5/6/7 induce the
ER stress response?

It remains therefore to be seen if the expression of PRRSV proteins increases the formation of autophagosomes and/or autophagy-like vesicles similar to the coronaviral nsp-3/-4/-6 proteins whilst inhibiting the fusion of the lysosome. Also, it remains to be seen if the expression of PRRSV nsp-2 -and other viral proteins including nsp-5/6/7 induces the ER stress response by lipid depletion and subsequent autosis. Interestingly the infection of MARC-145 cells with PRRSV strain CH-1a results in a PERK and IRE1 induced ER stress response whose inhibition is associated with decreased viral replication. Since autophagy is induced fooling the activation of the ER stress response it seems possible that the decrease in viral replication is due to a decrease in autophagy or alternatively to apoptosis (autophagy dependent or independent). In this case, the activation of the ER stress response might induce p53 and thus DRAM-1; indeed, the inhibition of p53 has been demonstrated to decrease viral titers, but so far no link has been established between PRRSV, the ER stress response, p53, and autophagy.  

Interplay of the induction of Akt, Akt dependent inhibition of autophagy and induction
of autophagy via the ER stress response: activation of Akt early during the infection, induction of the ER
stress response late in infection?

Further reading

Meulenberg, J. (2000). PRRSV, the virus Veterinary Research, 31 (1), 11-21 DOI: 10.1051/vetres:2000103 

Sun Z, Chen Z, Lawson SR, & Fang Y (2010). The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions. Journal of virology, 84 (15), 7832-46 PMID: 20504922 

Shi X, Zhang G, Wang L, Li X, Zhi Y, Wang F, Fan J, & Deng R (2011). The nonstructural protein 1 papain-like cysteine protease was necessary for porcine reproductive and respiratory syndrome virus nonstructural protein 1 to inhibit interferon-β induction. DNA and cell biology, 30 (6), 355-62 PMID: 21438756 

You JH, Howell G, Pattnaik AK, Osorio FA, & Hiscox JA (2008). A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging. Virology, 378 (1), 34-47 PMID: 18550142

Chen Z, Zhou X, Lunney JK, Lawson S, Sun Z, Brown E, Christopher-Hennings J, Knudsen D, Nelson E, & Fang Y (2010). Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response. The Journal of general virology, 91 (Pt 4), 1047-57 PMID: 19923257

Fang Y, Treffers EE, Li Y, Tas A, Sun Z, van der Meer Y, de Ru AH, van Veelen PA, Atkins JF, Snijder EJ, & Firth AE (2012). Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein. Proceedings of the National Academy of Sciences of the United States of America, 109 (43) PMID: 23043113 

Snijder EJ, van Tol H, Pedersen KW, Raamsman MJ, & de Vries AA (1999). Identification of a novel structural protein of arteriviruses. Journal of virology, 73 (8), 6335-45 PMID: 10400725 

Oleksiewicz MB, & Nielsen J (1999). Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function. Veterinary microbiology, 66 (1), 15-27 PMID: 10223319 

 Monastyrska I, Ulasli M, Rottier PJ, Guan JL, Reggiori F, & de Haan CA (2013). An autophagy-independent role for LC3 in equine arteritis virus replication. Autophagy, 9 (2), 164-74 PMID: 23182945 

Knoops K, Bárcena M, Limpens RW, Koster AJ, Mommaas AM, & Snijder EJ (2012). Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. Journal of virology, 86 (5), 2474-87 PMID: 22190716 

Lu W, Sun B, Mo J, Zeng X, Zhang G, Wang L, Zhou Q, Zhu L, Li Z, Xie Q, Bi Y, & Ma J (2014). Attenuation and immunogenicity of a live high pathogenic PRRSV vaccine candidate with a 32-amino acid deletion in the nsp2 protein. Journal of immunology research, 2014 PMID: 25009824

Pujhari S, Kryworuchko M, & Zakhartchouk AN (2014). Role of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) signalling pathways in porcine reproductive and respiratory syndrome virus (PRRSV) replication. Virus research, 194, 138-44 PMID: 25304692

Cottam EM, Whelband MC, & Wileman T (2014). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10 (8), 1426-41 PMID: 24991833 S

Sun MX, Huang L, Wang R, Yu YL, Li C, Li PP, Hu XC, Hao HP, Ishag HA, & Mao X (2012). Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication. Autophagy, 8 (10), 1434-47 PMID: 22739997 

Liu Q, Qin Y, Zhou L, Kou Q, Guo X, Ge X, Yang H, & Hu H (2012). Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells. Virology, 429 (2), 136-47 PMID: 22564420 

Chen Q, Fang L, Wang D, Wang S, Li P, Li M, Luo R, Chen H, & Xiao S (2012). Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication. Virus research, 163 (2), 650-5 PMID: 22119900 

Cottam EM, Maier HJ, Manifava M, Vaux LC, Chandra-Schoenfelder P, Gerner W, Britton P, Ktistakis NT, & Wileman T (2011). Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy, 7 (11), 1335-47 PMID: 21799305 

Huo Y, Fan L, Yin S, Dong Y, Guo X, Yang H, & Hu H (2013). Involvement of unfolded protein response, p53 and Akt in modulation of porcine reproductive and respiratory syndrome virus-mediated JNK activation. Virology, 444 (1-2), 233-40 PMID: 23850458

Muñoz-Pinedo, C., & Martin, S. (2014). Autosis: a new addition to the cell death tower of babel Cell Death and Disease, 5 (7) DOI: 10.1038/cddis.2014.246 

Liu Y, Shoji-Kawata S, Sumpter RM Jr, Wei Y, Ginet V, Zhang L, Posner B, Tran KA, Green DR, Xavier RJ, Shaw SY, Clarke PG, Puyal J, & Levine B (2013). Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proceedings of the National Academy of Sciences of the United States of America, 110 (51), 20364-71 PMID: 24277826

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